Co-enzyme Q10 formulations and methods of use

ABSTRACT

Topical formulations of CoQ10 reduce the rate of tumor growth in an animal subject. In the experiments described herein, CoQ10 was shown to increase the rate of apoptosis in a culture of skin cancer cells but not normal cells. Moreover, treatment of tumor-bearing animals with a topical formulation of CoQ10 was shown to dramatically reduce the rate of tumor growth in the animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/366,224, filed Feb. 3, 2012; which is a continuation of U.S.application Ser. No. 10/597,378, filed Aug. 21, 2008, now granted asU.S. Pat. No. 8,147,825; which is a 371 of International ApplicationPCT/US2005/001581, filed Jan. 21, 2005, which, in turn, claims priorityunder 35 U.S.C. §119 from U.S. Application Ser. No. 60/538,319, filedJan. 22, 2004. The entire contents of each of the foregoing applicationsand patents are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention provides pharmaceutical compositions comprising co-enzymeQ10 (CoQ10) and methods of using CoQ10 for treatment of cancer,selective reduction of cancer cell growth, induction of apoptosis incancer cells and inhibition of tumor mediated angiogenesis.

BACKGROUND

Cancer is presently one of the leading causes of death in developednations. Although recent research has vastly increased our understandingof many of the molecular mechanisms of tumorigenesis and has providednumerous new avenues for the treatment of cancer, standard treatmentsfor most malignancies remain gross resection, chemotherapy, andradiotherapy. While increasingly successful, each of these treatmentsstill causes numerous undesired side effects. For example, surgeryresults in pain, traumatic injury to healthy tissue, and scarring.Radiotherapy and chemotherapy cause nausea, immune suppression, gastriculceration and secondary tumorigenesis.

SUMMARY

The invention relates to the discovery that topical formulations ofCoQ10 can reduce the rate of tumor growth in an animal subject. In theexperiments described herein, CoQ10 was shown to increase the rate ofapoptosis in a culture of skin cancer cells but not normal cells.Moreover, treatment of tumor-bearing animals with a topical formulationof CoQ10 was shown to dramatically reduce the rate of tumor growth inthe animals.

CoQ10 formulated for oral delivery has previously been used as a dietarysupplement. Orally administered CoQ10 has, however, been shown toaccumulate in the liver-diminishing its systemic availability. Theanti-tumor responses observed with topically applied CoQ10 may relate toits higher bioavailability compared to dietary supplement forms of theCoQ10.

Accordingly, the invention features a method for reducing the rate oftumor cell growth or increasing the rate of apoptosis in tumor cells ina subject. The method includes the steps of providing a subject having aplurality of tumor cells and administering to the subject a compositioncomprising an effective amount of CoQ10 and a pharmaceuticallyacceptable carrier.

In another aspect, the invention features a composition comprising aneffective amount of CoQ10 and a pharmaceutically acceptable carrier.

In a preferred embodiment, the composition is a topical formulation ofCoQ10 that includes at least about 0.01% by weight CoQ10 up to 30% byweight (w/w) of CoQ10 and a carrier suitable for delivering the CoQ10topically. Preferably, the pharmaceutical composition comprises as anactive ingredient CoQ10 and a pharmaceutically acceptable carrier. Thecomposition comprising, Coenzyme Q10, phospholipon 90, glycerol,butylated hydroxytoluene (BHT), ethanol, medium chain triglycerides(MCT) and lavender. Preferably, the phospholipon 90 is phospholipon 90Gand/or phospholipon 90H.

In a preferred embodiment, the pharmaceutical composition comprises atleast about 0.01% to about 30% (w/w) of Coenzyme Q10. Preferably, thepharmaceutical composition between about 1% to about 25% (w/w) ofCoenzyme Q10.

In another preferred embodiment, the invention provides a method oftreating a cancer patient, comprising:

-   -   administering to a patient in need thereof, a composition        comprising a therapeutically effective amount of Coenzyme Q10;    -   contacting a tumor cell with the composition resulting in the        lysis of the tumor cell; thereby treating the cancer patient.        Preferably, the pharmaceutical composition comprises at least        about 0.01% up to 30% w/w of Coenzyme Q10, preferably, the        pharmaceutical composition comprises about 1% to about 25% w/w        of Coenzyme Q10.

In another preferred embodiment, the pharmaceutical composition isformulated in a topical cream with optional transdermal enhancers.

In other preferred embodiments, a therapeutically effective amount ofthe Coenzyme Q10 composition is administered with one or morechemotherapeutic agents. These chemotherapeutic agents can beco-administered, precede, or administered after the Coenzyme Q10.Non-limiting examples of chemotherapeutic agents include, but notlimited to: cyclophosphamide (CTX, 25 mg/kg/day, p.o.), taxanes(paclitaxel or docetaxel), busulfan, cisplatin, cyclophosphamide,methotrexate, daunorubicin, doxorubicin, melphalan, cladribine,vincristine, vinblastine, and chlorambucil.

In another preferred embodiment, the pharmaceutical composition,Coenzyme Q10 composition inhibits the tumor cell growth in a subject,and the method comprises administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of CoQ10.Preferably, the therapeutically effective amount of Coenzyme Q10 in thepharmaceutical composition comprises between about 0.01% and 30% w/w ofcoenzyme Q10. Inhibition of tumor cell growth refers to one or more ofthe following effects: (1) inhibition, to some extent, of tumor growth,including, (i) slowing down and (ii) complete growth arrest; (2)reduction in the number of tumor cells; (3) maintaining tumor size; (4)reduction in tumor size; (5) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of tumor cell infiltrationinto peripheral organs; (6) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of metastasis; (7)enhancement of anti-tumor immune response, which may result in (i)maintaining tumor size, (ii) reducing tumor size, (iii) slowing thegrowth of a tumor, (iv) reducing, slowing or preventing invasion and/or(8) relief, to some extent, of the severity or number of one or moresymptoms associated with the disorder.

In another preferred embodiment, the invention provides a method ofinducing apoptosis selectively in a tumor cell, the method comprisingadministering a pharmaceutical composition comprising coenzyme Q10 asmeasured in standard assays. Preferably, the pharmaceutical compositioncomprises at least about 0.01% up to 30% w/w of Coenzyme Q10. Methodsfor measuring apoptosis include but not limited to mitochondrialmembrane dye assays and/or Annexin-VPE assays. In a preferredembodiment, the pharmaceutical composition induces apoptosis in at leastabout 30% of tumor cells as measured by mitochondrial membrane dye assayand/or Annexin-VPE assay. Preferably, the pharmaceutical compositioninduces apoptosis in about 60% of tumor cells as measured bymitochondrial membrane dye assay and/or Annexin-VPE assay, morepreferably, the pharmaceutical composition induces apoptosis in about75% of tumor cells as measured by mitochondrial membrane dye assayand/or Annexin-VPE assay, more preferably, the pharmaceuticalcomposition induces apoptosis in about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% and 100% of tumor cells as measured by mitochondrialmembrane dye assay and/or Annexin-VPE assay.

In another preferred embodiment, the invention provides a method ofinhibiting angiogenesis in a tumor, the method comprising contacting atumor with a pharmaceutical composition comprising coenzyme Q10.Preferably, the pharmaceutical composition comprises at least about0.01% up to 30% w/w of Coenzyme Q10.

Additional uses for the present compounds include use in the treatmentof atherosclerosis, inflammation, and as an anti-angiogenic agent,especially to treat cancers, particularly solid cancers such as cancersresiding in the lung, breast, liver, brain or other tissue.

Unless otherwise defined, all technical terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions of medicalterms can be found in Thomas Lathrop Stedman, Stedman's MedicalDictionary, Lippincott, Williams & Wilkins: Philadelphia, Pa., 2000.

All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. The particular embodiments discussed below are illustrativeonly and not intended to be limiting.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is a series of photomicrographs showing the effect of CoQ10 onhuman melanoma cells (SKMEL28) in an in vitro culture.

FIG. 2 is a graph showing that CoQ10 reduces the proliferation of ahuman melanoma cell line (SKMEL28) in a 36 hour in vitro culture.

FIG. 3 is a graph showing that CoQ10 reduces the proliferation of ahuman melanoma cell line (SKMEL28) in a 48 hour in vitro culture.

FIG. 4 is a graph showing that the vehicle control does not reduce theproliferation of a human melanoma cell line (SKMEL28) in a 48 hour invitro culture.

FIG. 5 is a graph comparing the effect of CoQ10 on apoptosis betweenhuman melanoma and neonatal fibroblasts in an in vitro culture.

FIG. 6 is a graph showing that CoQ10 reduces the proliferation ofsquamous carcinoma cells in a 48 hour in vitro culture.

FIG. 7 is a graph showing that CoQ10 increases the proliferation ofhuman neonatal fibroblasts in a 48 hour in vitro culture.

FIG. 8 is a graph showing that CoQ10 increases the proliferation ofhuman neonatal keratinocytes in a 48 hour in vitro culture.

FIG. 9 is a graph showing that CoQ10 reduces the proliferation of abreast adenocarcinoma cell line (MCF-7) in a 48 hour in vitro culture.The MCF-7 cell line expresses the WNT7B oncogene and contains the Tx-4oncogene.

FIG. 10 is a graph showing that CoQ10 reduces the proliferation of abreast adenocarcinoma cell line (MCF-7) in a 72 hour in vitro culture.

FIG. 11 is a photograph showing induced tumors in control andCoQ10-treated mice after treatment with topical formulation of CoQ10 for30 days.

FIG. 12 is a photograph showing induced tumors in control andCoQ10-treated mice after treatment with topical formulation of CoQ10 for30 days.

FIG. 13 is a photograph showing tumors excised from control andCoQ10-treated mice.

FIG. 14 is a graph showing the effect of CoQ10 administration on tumorsize on mice treated topically with CoQ10 (1.0% or 1.5%) or control for30 days. Average tumor mass in the 1.0% CoQ10 and 1.5% CoQ10 treatmentgroups decreased by 52.3% and 54.0%, respectively, as compared to thecontrol.

FIG. 15 is a series of photomicrographs showing the effect of CoQ10 onhuman breast adenocarcinoma cells (SK-BR-3) in an in vitro culture. TheSK-BR-3 cell line overexpresses the Her2/c-erb-2 genes, gene product(ATCC).

FIG. 16 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (SK-BR-3) in a 48 hour in vitroculture. The SK-BR-3 cell line overexpresses the Her2/c-erb-2 genes geneproduct (ATCC).

FIG. 17 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (SK-BR-3) in a 72 hour in vitroculture. The SK-BR-3 cell line overexpresses the Her2/c-erb-2 genes geneproduct (ATCC).

FIG. 18 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (MDA-MB-468) in a 48 hour in vitroculture. The MDA-MB-468 cell line has a mutation in the p53 gene (ATCC).

FIG. 19 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (MDA-MB-468) in a 72 hour in vitroculture. The MDA-MB-468 cell line has a mutation in the p53 gene (ATCC).

FIG. 20 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (BT-20) in a 48 hour in vitroculture. The BT-20 cell line expresses the WNT7B and WNT3 oncogene(ATCC).

FIG. 21 is a graph showing that CoQ10 reduces the proliferation of ahuman breast adenocarcinoma cell line (BT-20) in a 72 hour in vitroculture. The BT-20 cell line expresses the WNT7B and WNT3 oncogene(ATCC).

FIG. 22 is a graph showing that CoQ10 reduces the proliferation of ahuman hepatocellular carcinoma cell line (Hep 3B) in a 48 hour in vitroculture.

FIG. 23 is a graph showing that CoQ10 reduces the proliferation of ahuman hepatocellular carcinoma cell line (Hep 3B) in a 72 hour in vitroculture.

FIG. 24 is a graph showing that CoQ10 reduces the proliferation of ahuman osteosarcoma cell line (143B) in a 48 hour in vitro culture.

FIG. 25 is a graph showing that CoQ10 reduces the proliferation of ahuman osteosarcoma cell line (143B) in a 72 hour in vitro culture.

FIG. 26 is a graph showing that CoQ10 reduces the proliferation of ahuman prostatic adenocarcinoma cell line (PC-3) in a 48 hour in vitroculture.

FIG. 27 is a graph showing that CoQ10 reduces the proliferation of ahuman prostatic adenocarcinoma cell line (PC-3) in a 72 hour in vitroculture.

FIG. 28 is a graph showing the effect of CoQ10 on mitochondrialpolarization (an indicator of apoptosis) of a human prostaticadenocarcinoma cell line (PC-3) in a 24 hour in vitro culture. PC-3 cellcultures were treated with Q10 at 0.05, 0.1 and 0.2 mM concentrationsfor 24 h and then treated with JC-1, at 10 microgram/ml., for 30 min.Uptake and levels of green fluorescence was measured in a flowcytometer, FL1 (green fluor.). Note: A significant increase in the greenfluorescence was observed in 0.2 mM Q10 treated cells (yellow graph).

FIGS. 29A and 29B are photographs showing inhibition of tumor-mediatedangiogenesis in tissues by a composition comprising CoQ10 (FIG. 29B) ascompared to a control in the absence of a composition comprising CoQ10.

DETAILED DESCRIPTION

The invention provides compositions and methods for reducing the rate oftumor cell growth or increasing the rate of tumor cell apoptosis.Compositions of the invention include as an anti-tumor agent atherapeutically effective amount of CoQ10 and a carrier. A preferredcomposition of the invention is a topical formulation of CoQ10comprising at least about 1% CoQ10 and a carrier that facilitatestopical delivery of CoQ10. A most preferred composition of the inventionis a topical formulation of CoQ10 comprising between about 1% and 15%CoQ10 and a carrier that facilitates topical delivery of CoQ10. Methodsof the invention for killing a tumor cell or reducing its growth rateinclude the step of contacting the cell with an effective concentrationof CoQ10.

The below described preferred embodiments illustrate adaptations ofthese compositions and methods. Nonetheless, from the description ofthese embodiments, other aspects of the invention can be made and/orpracticed based on the description provided below.

Before the present invention is disclosed and described, it is to beunderstood that this invention is not limited to the particularstructures, process steps, or materials disclosed herein, but isextended to equivalents thereof as would be recognized by thoseordinarily skilled in the relevant arts. It should also be understoodthat terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

As used herein, “a”, “an,” and “the” include plural references unlessthe context clearly dictates otherwise.

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio.

As used herein, the term “safe and therapeutic effective amount” refersto the quantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example, an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, or to shrink the cancer orprevent metastasis. The specific safe and effective amount ortherapeutically effective amount will vary with such factors as theparticular condition being treated, the physical condition of thepatient, the type of mammal or animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives.

As used herein, a “pharmaceutical salt” include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids. Preferablythe salts are made using an organic or inorganic acid. These preferredacid salts are chlorides, bromides, sulfates, nitrates, phosphates,sulfonates, formates, tartrates, maleates, malates, citrates, benzoates,salicylates, ascorbates, and the like. The most preferred salt is thehydrochloride salt.

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. In preferredembodiments, the CoQ10 compositions are used for treatment, of varioustypes of breast cancer; prostate cancer; liver cancer; bone cancer.However, treatment using the CoQ10 compositions are not limited to thesetypes of cancers.

Examples of cancers are cancer of the brain, breast, pancreas, cervix,colon, head and neck, kidney, lung, non-small cell lung, melanoma,mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma. Asused herein, the terms “cancer,” “neoplasm,” and “tumor,” are usedinterchangeably and in either the singular or plural form, refer tocells that have undergone a malignant transformation that makes thempathological to the host organism. Primary cancer cells (that is, cellsobtained from near the site of malignant transformation) can be readilydistinguished from non-cancerous cells by well-established techniques,particularly histological examination. The definition of a cancer cell,as used herein, includes not only a primary cancer cell, but any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by procedures such as CAT scan, MR imaging,X-ray, ultrasound or palpation, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sampleobtainable from a patient.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Examples of sarcomas which can be treated with the presentcompositions and optionally a potentiator and/or chemotherapeutic agentinclude, but not limited to a chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with the compositions of the invention and optionally apotentiator and/or another chemotherapeutic agent include but notlimited to, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, and superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Carcinomas which can be treated with thecompositions of the invention and optionally a potentiator and/or achemotherapeutic agent include but not limited to, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma an cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant call carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma apongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telagiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

Additional cancers which can be treated with the compositions of theinvention include, for example, Hodgkin's Disease, Non-Hodgkin'sLymphoma, multiple myeloma, neuroblastoma, breast cancer, ovariancancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumors, primary brain tumors, stomachcancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal cortical cancer, and prostate cancer.

“Diagnostic” or “diagnosed” means identifying the presence or nature ofa pathologic condition. Diagnostic methods differ in their sensitivityand specificity. The “sensitivity” of a diagnostic assay is thepercentage of diseased individuals who test positive (percent of “truepositives”). Diseased individuals not detected by the assay are “falsenegatives.” Subjects who are not diseased and who test negative in theassay, are termed “true negatives.” The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the “false positive”rate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

The terms “patient” or “individual” are used interchangeably herein, andrefers to a mammalian subject to be treated, with human patients beingpreferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary application, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; and primates.

“Sample” is used herein in its broadest sense. A sample comprisingpolynucleotides, polypeptides, peptides, antibodies and the like maycomprise a bodily fluid; a soluble fraction of a cell preparation, ormedia in which cells were grown; a chromosome, an organelle, or membraneisolated or extracted from a cell; genomic DNA, RNA, or cDNA,polypeptides, or peptides in solution or bound to a substrate; a cell; atissue; a tissue print; a fingerprint, skin or hair; and the like.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. As used herein,“ameliorated” or “treatment” refers to a symptom which is approaches anormalized value (for example a value obtained in a healthy patient orindividual), e.g., is less than 50% different from a normalized value,preferably is less than about 25% different from a normalized value,more preferably, is less than 10% different from a normalized value, andstill more preferably, is not significantly different from a normalizedvalue as determined using routine statistical tests. For example, the“treatment of cancer” or “tumor cells”, refers to one or more of thefollowing effects: (1) inhibition, to some extent, of tumor growth,including, (i) slowing down and (ii) complete growth arrest; (2)reduction in the number of tumor cells; (3) maintaining tumor size; (4)reduction in tumor size; (5) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of tumor cell infiltrationinto peripheral organs; (6) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of metastasis; (7)enhancement of anti-tumor immune response, which may result in (i)maintaining tumor size, (ii) reducing tumor size, (iii) slowing thegrowth of a tumor, (iv) reducing, slowing or preventing invasion and/or(8) relief, to some extent, of the severity or number of one or moresymptoms associated with the disorder.

As used herein, “an ameliorated symptom” or “treated symptom” refers toa symptom which is approaches a normalized value, e.g., is less than 50%different from a normalized value, preferably is less than about 25%different from a normalized value, more preferably, is less than 10%different from a normalized value, and still more preferably, is notsignificantly different from a normalized value as determined usingroutine statistical tests.

A “chemokine” is a small cytokine involved in the migration andactivation of cells, including phagocytes and lymphocytes, and plays arole in inflammatory responses.

A “cytokine” is a protein made by a cell that affect the behavior ofother cells through a “cytokine receptor” on the surface of the cellsthe cytokine effects. Cytokines manufactured by lymphocytes aresometimes termed “lymphokines.” Cytokines are also characterized as TypeI (e.g. IL-2 and IFN-γ) and Type II (e.g. IL-4 and IL-10).

By the term “modulate,” it is meant that any of the mentionedactivities, are, e.g., increased, enhanced, increased, agonized (acts asan agonist), promoted, decreased, reduced, suppressed blocked, orantagonized (acts as an agonist). Modulation can increase activity morethan 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., overbaseline values. Modulation can also decrease its activity belowbaseline values.

As used herein, the term “selective for tumor cells” refers to theeffects of the Coenzyme Q10 pharmaceutical compositions, such asinhibition of tumor growth, apoptosis, anti-angiogenic effects and whichare not detectable when applied to normal cells, as described in detailin the examples which follow.

CoQ10 Compositions

In a preferred embodiment, the invention provides CoQ10 compositions forthe treatment of cancer. Preferably, the compositions comprise at leastabout 1% to about 25% CoQ10 w/w, more preferably, between about 1% toabout 20% CoQ10 w/w. In the representative embodiment described in theExamples section below, a topical formulation of CoQ10 is applied to theskin of a tumor-bearing animal to reduce the growth rate of the tumor.CoQ10 can be obtained from Pure Prescriptions (San Diego, Calif.) inpowdered form in any suitable quantity (e.g., 1 kilogram). To deliver aCoQ10-containing composition, any suitable carrier can be used.Liposomes, for example, may be used as a carrier. An exemplary liposomalformulation is composed of Phospholipon 90G (American Lechitin,Stanford, Conn.), Phospholipon 90H (American Lechitin, Stanford, Conn.),Glycerol, Butylated hydroxytoluene (BHT), Ethanol, Medium ChainTriglycerides (MCT), lavender (Sigma-Aldrich, St. Louis, MO.) andCoenzyme Q10 (Pure Prescriptions, San Diego, CA). An example of aprotocol for preparing this formulation entails first dissolving 10 g ofPhospholipon 90H, 5 g Phospholipon 90G, with 1.5 g MCT, 0.3 g BHT, and 9ml of ethanol at 75° C. Next, 1.1 g of Coenzyme Q10 are dissolved intothe mixture, 65 ml of 1 mM phosphate buffer (pH 8.2) prepared withnitrogen saturated water, 13.3 g glycerol, and 50 μL of lavender areadded. The above mixture is blended in a high-speed blender at 12,000RPM to form a cream. The cream is stored at 4° C. until used.

Subjects

Because subjects from many different species have tumors and aresusceptible to acquiring a tumor, the invention is compatible with manytypes of animal subjects. A non-exhaustive exemplary list of suchanimals includes mammals such as mice, rats, rabbits, goats, sheep,pigs, horses, cattle, dogs, cats, and primates such as monkeys, apes,and human beings. Those animal subjects known to suffer from a skincancer tumor are preferred for use in the invention. In particular,human patients suffering from a skin cancer tumor or other tumors aresuitable animal subjects for use in the invention. By adapting themethods taught herein to other methods known in medicine or veterinaryscience (e.g., adjusting doses of administered substances according tothe weight of the subject animal), the compositions utilized in theinvention can be readily optimized for use in other animals.

Pharmaceutical Compositions and Administration to a Subject

In a preferred embodiment, the compositions comprising CoQ10 areadministered topically. It is preferable to present the activeingredient, i.e. CoQ10 as a pharmaceutical formulation. Exemplarycompositions are described in detail in the examples which follow. Theactive ingredient may comprise, for topical administration, from 0.001%to about 20% w/w, by weight of the formulation in the final product.,although it may comprise as much as 30% w/w, preferably from about 1% toabout 20% w/w of the formulation. The topical formulations of thepresent invention, comprise an active ingredient together with one ormore acceptable carrier(s) therefor and optionally any other therapeuticingredients(s). The carrier(s) must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The composition of the invention can be administered to a patient eitherby themselves, or in pharmaceutical compositions where it is mixed withsuitable carriers or excipient(s). In treating a patient exhibiting adisorder of interest, a therapeutically effective amount of a agent oragents such as these is administered. A therapeutically effective doserefers to that amount of the compound that results in amelioration ofsymptoms or a prolongation of survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oncogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

The compositions described above may be administered to a subject in anysuitable formulation. In addition to treatment of cancer with topicalformulations of CoQ10, in other aspects of the invention CoQ10 might bedelivered by other methods. For example, CoQ10 might be formulated forparenteral delivery, e.g., for subcutaneous, intravenous, intramuscular,or intratumoral injection. Other methods of delivery, for example,liposomal delivery or diffusion from a device impregnated with thecomposition might be used. The compositions may be administered in asingle bolus, multiple injections, or by continuous infusion (forexample, intravenously or by peritoneal dialysis). For parenteraladministration, the compositions are preferably formulated in asterilized pyrogen-free form. Compositions of the invention can also beadministered in vitro to a cell (for example, to induce apoptosis in acancer cell in an in vitro culture) by simply adding the composition tothe fluid in which the cell is contained.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable mutes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. See, for example, FIG.14. Determination of the effective amounts is well within the capabilityof those skilled in the art, especially in light of the detaileddisclosure provided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration may be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions of the present invention maybe manufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes, Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coating. For this purpose,concentrated sugar solutions may be used, which may optionally containgun arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

The composition can include a buffer system, if desired. Buffer systemsare chosen to maintain or buffer the pH of compositions within a desiredrange. The term “buffer system” or “buffer” as used herein refers to asolute agent or agents which, when in a water solution, stabilize suchsolution against a major change in pH (or hydrogen ion concentration oractivity) when acids or bases are added thereto. Solute agent or agentswhich are thus responsible for a resistance or change in pH from astarting buffered pH value in the range indicated above are well known.While there are countless suitable buffers, potassium phosphatemonohydrate is a preferred buffer.

The final pH value of the pharmaceutical composition may vary within thephysiological compatible range. Necessarily, the final pH value is onenot irritating to human skin and preferably such that transdermaltransport of the active compound, i.e. CoQ10 is facilitated. Withoutviolating this constraint, the pH may be selected to improve CoQ10compound stability and to adjust consistency when required. In oneembodiment, the preferred pH value is about 3.0 to about 7.4, morepreferably about 3.0 to about 6.5, most preferably from about 3.5 toabout 6.0.

For preferred topical delivery vehicles the remaining component of thecomposition is water, which is necessarily purified, e.g., deionizedwater. Such delivery vehicle compositions contain water in the range ofmore than about 50 to about 95 percent, based on the total weight of thecomposition. The specific amount of water present is not critical,however, being adjustable to obtain the desired viscosity (usually about50 cps to about 10,000 cps) and/or concentration of the othercomponents. The topical delivery vehicle preferably has a viscosity ofat least about 30 centipoises.

Other known transdermal skin penetration enhancers can also be used tofacilitate delivery of CoQ10. Illustrative are sulfoxides such asdimethylsulfoxide (DMSO) and the like; cyclic amides such as1-dodecylazacycloheptane-2-one (Azone™, a registered trademark of NelsonResearch, Inc.) and the like; amides such as N,N-dimethyl acetamide(DMA) N,N-diethyl toluamide, N,N-dimethyl formamide, N,N-dimethyloctamide, N,N-dimethyl decamide, and the like; pyrrolidono derivativessuch as N-methyl-2-pyrrolidone, 2-pyrrolidone,2-pyrrolidone-5-carboxylic acid, N-(2-hydroxyethyl)-2-pyrrolidone orfatty acid esters thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,N-tallowalkylpyrrolidones, and the like; polyols such as propyleneglycol, ethylene glycol, polyethylene glycol, dipropylene glycol,glycerol, hexanetriol, and the like; linear and branched fatty acidssuch as oleic, linoleic, lauric, valeric, heptanoic, caproic, myristic,isovaleric, neopentanoic, trimethyl hexanoic, isostearic, and the like;alcohols such as ethanol, propanol, butanol, octanol, oleyl, stearyl,linoleyl, and the like; anionic surfactants such as sodium laurate,sodium lauryl sulfate, and the like; cationic surfactants such asbenzalkonium chloride, dodecyltrimethylammonium chloride,cetyltrimethylammonium bromide, and the like; non-ionic surfactants suchas the propoxylated polyoxyethylene ethers, e.g., Poloxamer 231,Poloxamer 182, Poloxamer 184, and the like, the ethoxylated fatty acids,e.g., Tween 20, Myrj 45, and the like, the sorbitan derivatives, e.g.,Tween 40, Tween 60, Tween 80, Span 60, and the like, the ethoxylatedalcohols, e.g., polyoxyethylene (4) lauryl ether (Brij 30),polyoxyethylene (2) oleyl ether (Brij 93), and the like, lecithin andlecithin derivatives, and the like; the terpenes such as D-limonene,α-pinene, β-carene, α-terpineol, carvol, carvone, menthone, limoneneoxide, α-pinene oxide, eucalyptus oil, and the like.

Also suitable as skin penetration enhancers are organic acids and esterssuch as salicyclic acid, methyl salicylate, citric acid, succinic acid,and the like.

Angiogenesis and Angiogenesis-Dependent Diseases

As used herein, the terms “angiogenesis inhibitory”, “angiogenesisinhibiting” or “anti-angiogenic” include vasculogenesis, and areintended to mean effecting a decrease in the extent, amount, or rate ofneovascularization. Effecting a decrease in the extent, amount, or rateof endothelial cell proliferation or migration in the tissue is aspecific example of inhibiting angiogenesis.

The term “angiogenesis inhibitory composition” refers to a compositioncomprising CoQ10 which inhibits tumor mediated angiogenesis processessuch as endothelial cell migration, proliferation, tube formation andsubsequently leading to the inhibition of the generation of new bloodvessels from existing ones, and consequently the inhibition ofangiogenesis-dependent diseases, for example, angiogenesis mediated bytumors. See, for example FIGS. 29A and 29B wherein a compositioncomprising CoQ10 inhibits tumor-mediated angiogenesis in a tissue ascompared to control tissue in the absence of any CoQ10. The compositioncomprising CoQ10 is described in detail in the examples which follow.

As used herein, the term “angiogenesis-dependent disease” is intended tomean a disease where the process of angiogenesis or vasculogenesissustains or augments a pathological condition. In particular,angiogenesis-dependent disease refers to tumor-mediated angiogenesis.

Angiogenesis is the formation of new blood vessels from pre-existingcapillaries or post-capillary venules. Vasculogenesis results from theformation of new blood vessels arising from angioblasts which areendothelial cell precursors. Both processes result in new blood vesselformation and are included in the meaning of the termangiogenesis-dependent diseases. Similarly, the term “angiogenesis” asused herein is intended to include de novo formation of vessels such asthat arising from vasculogenesis as well as those arising from branchingand sprouting of existing vessels, capillaries and venules.

Angiogeniesis, including vasculogenesis, is an important physiologicalprocess, without which embryonic development and wound healing would notoccur. However, angiogenesis is also inappropriately recruited intonumerous pathological conditions as a means to provide adequate bloodand nutrient supply to the cells within the affected tissue. Many ofthese pathological conditions involve aberrant cell proliferation orregulation. Such conditions in which angiogenesis is believed to beimportant are referred to herein as angiogenesis-dependent diseases.However, methods of the invention also can be used beneficially toinhibit angiogenesis associated with normal physiological processes. Forexample, the inhibition of angiogenesis associated with the menstrualcycle can be prophylactically used as an effective method of birthcontrol. Therefore, the description below in reference to the treatmentof angiogenesis-dependent diseases are also applicable to the inhibitionof normal angiogenic responses where a prophylactic or therapeutic needor benefit exists.

Angiogenesis-dependent diseases include, for example, inflammatorydisorders such as immune and non-immune inflammation, rheumatoidarthritis, chronic articular rheumatism and psoriasis; disordersassociated with inappropriate or inopportune invasion of vessels such asdiabetic retinopathy, neovascular glaucoma, retinopathy of prematurity,macular degeneration, corneal graft rejection, retrolental fibroplasia,rubeosis, capillary proliferation in atherosclerotic plaques andosteoporosis; and cancer associated disorders, including for example,solid tumors, tumor metastases, blood born tumors such as leukemias,angiofibromas, Kaposi sarcoma, benign tumors such as hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, aswell as other cancers which require neovascularization to support tumorgrowth. Additional examples of angiogenesis-dependent diseases include,for example, Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints and woundgranulation. Other diseases in which angiogenesis plays a role in themaintenance or progression of the pathological state are known to thoseskilled in the art and are similarly intended to be included within themeaning of the term used herein. Preferably, angiogenesis-mediateddiseases refers to tumor induced angiogenesis.

In Vitro Biological Assay of Angiogenesis Inhibiting Activity

The CoQ10 compounds of the instant invention can be tested for theirangiogenesis inhibiting activity in several assay systems in vitro andare well within the knowledge of one of ordinary skill in the art.Endothelial cells, for example, human umbilical vein endothelial cells(HUVEC) or human microvascular endothelial cells (HMVEC) can be preparedor obtained commercially, are mixed at a concentration of 2×10⁵ cells/mLwith fibrinogen (5 mg/mL in phosphate buffered saline (PBS) in a 1:1(v/v) ratio. Thrombin is added (5 units/mL final concentration) and themixture immediately transferred to a 24-well plate (0.5 mL per well).The fibrin gel is allowed to form and then vascular endothelial growthfactor (VEGF) and fibroblast growth factor basic (FGF2) are added to thewells (each at 5 ng/mL, final concentration) along with the testcompound, as described in the Examples which follow. The cells areincubated at 37° C. in 5% CO₂ for 4 days at which time the cells in eachwell are counted and classified as either rounded, elongated with nobranches, elongated with one branch, or elongated with 2 or morebranches. Results are expressed as the average of 5 different wells foreach concentration of compound. Typically, in the presence of angiogenicinhibitors, cells remain either rounded or form undifferentiated tubes(e.g., 0 or 1 branch). This assay is recognized in the art to bepredictive of angiogenic efficacy (or angiogenesis inhibiting activity)In vivo (Grant et al., In Vitro Cell Dev. Biol. 27A:327-336 (1991); Minet al., Cancer Res. 56:2428-2433 (1996)).

In an alternate assay, endothelial cell tube formation is observed whenendothelial cells are cultured on Matrigel™ matrix-coated plates,commercially available from Becton Dickinson of Bedford, Pa. (Schnaperet al., J. Cell. Physiol. 165:107-118 (1995)). Endothelial cells (1×10⁴cells/well) are transferred onto Matrigel™ matrix-coated 24-well plates,and tube formation is quantitated after 48 hours. Inhibitors are testedby adding them either at the same time as the endothelial cells or atvarious time points thereafter.

This assay models angiogenesis by presenting to the endothelial cells aparticular type of basement membrane, namely the layer of matrix whichmigrating and differentiating endothelial cells might be expected tofirst encounter. In addition to bound growth factors, the matrixcomponents found in Matrigel™ matrix (and in basement membranes in situ)or proteolytic products thereof may also be stimulatory for endothelialcell tube formation which makes this model complementary to the fibringel angiogenesis model.

Additionally, angiogenic activities of compounds of the presentinvention can be evaluated by the chick chorioallantoic membrane (CAM)assay (Oikawa at al., Cancer Lett. 59:57-66 (1991)).

Combination Therapies

The CoQ10 therapeutic compositions of the present invention may becombined with any other methods generally employed in the treatment ofthe particular tumor, disease or disorder that the patient exhibits. Solong as a particular therapeutic approach is not known to be detrimentalto the patients condition in itself, and does not significantlycounteract the CoQ10 composition treatment, its combination with thepresent invention is contemplated.

In connection solid tumor treatment, the present invention may be usedin combination with classical approaches, such as surgery, radiotherapy,chemotherapy, and the like. The invention therefore provides combinedtherapies in which the CoQ10 therapeutic compositions are usedsimultaneously with, before, or after surgery or radiation treatment; orare administered to patients with, before, or after conventionalchemotherapeutic, radiotherapeutic or other anti-angiogenic agents, ortargeted immunotoxins or coaguligands.

Combination therapy for other vascular diseases is also contemplated. Aparticular example of such is benign prostatic hyperplasia (BPH), whichmay be treated with CoQ10 compositions in combination other treatmentscurrently practiced in the art. For example, targeting of immunotoxinsto markers localized within BPH, such as PSA.

When one or more agents are used in combination with the CoQ10compositions, there is no requirement for the combined results to beadditive of the effects observed when each treatment is conductedseparately. Although at least additive effects are generally desirable,any increased anti-tumor effect above one of the single therapies wouldbe of benefit. Also, there is no particular requirement for the combinedtreatment to exhibit synergistic effects, although this is certainlypossible and advantageous.

To practice combined anti-tumor therapy, one would simply administer toan animal a the CoQ10 composition construct in combination with anotheranti-cancer agent in a manner effective to result in their combinedanti-tumor actions within the animal. The agents would therefore beprovided in amounts effective and for periods of time effective toresult in their combined presence within the tumor vasculature and theircombined actions in the tumor environment. To achieve this goal, theCoQ10 compositions and other anti-cancer agents may be administered tothe animal simultaneously, either in a single composition, or as twodistinct compositions using different administration routes.

Alternatively, the CoQ10 composition mediated treatment may precede, orfollow, the a second anti-cancer agent treatment by, e.g., intervalsranging from minutes to weeks. In certain embodiments where theanti-cancer agent and the CoQ10 composition are applied separately tothe animal, one would ensure that a significant period of time did notexpire between the time of each delivery, such that the anti-canceragent and the CoQ10 composition would still be able to exert anadvantageously combined effect on the tumor. In such instances, it iscontemplated that one would contact the tumor with both agents withinabout 5 minutes to about one week of each other and, more preferably,within about 12-72 hours of each other, with a delay time of only about12-48 hours being most preferred.

The general use of combinations of substances in cancer treatment iswell known. For example, U.S. Pat. No. 5,710,134 (incorporated herein byreference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxio“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

In some situations, it may even be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as the CoQ10 compositiontreatment, and another treatment was intended to prevent micrometastasisor tumor re-growth, such as the administration of an anti-angiogenicagent.

It also is envisioned that more than one administration of either theCoQ10 composition or another anti-cancer agent will be utilized. TheCoQ10 composition and anti-cancer agents may be administeredinterchangeably, on alternate days or weeks; or a sequence of the CoQ10composition treatment may be given, followed by a sequence ofanti-cancer agent therapy. In any event, to achieve tumor regressionusing a combined therapy, all that is required is to deliver both agentsin a combined amount effective to exert an anti-tumor effect,irrespective of the times for administration.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within tumor cells iscontemplated, such as γ-irradiation, X-rays, UV-irradiation, microwavesand even electronic emissions and the like. The directed delivery ofradioisotopes to tumor cells is also contemplated, and this may be usedin connection with a targeting antibody or other targeting means.

Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α, TL-1β, IL-2, IL-3,IL-4, IL-5, TL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β,GM-CSF, M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP,LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administeredaccording to standard regimens, consistent with clinical indicationssuch as the condition of the patient and relative toxicity of thecytokine. Uteroglobins may also be used to prevent or inhibit metastases(U.S. Pat. No. 5,696,092; incorporated herein by reference).

CoQ10 Compositions and Combination Chemotherapeutics

In certain embodiments, the CoQ10 composition of the present inventionmay be administered in combination with another chemotherapeutic agent.Irrespective of the underlying mechanism(s), a variety ofchemotherapeutic agents may be used in the combined treatment methodsdisclosed herein. Therapeutic agents can include, for example,chemotherapeutic agents such as, cyclophosphamide (CTX, 25 mg/kg/day,p.o.), taxanes (paclitaxel or docetaxel), busulfan, cisplatin,methotrexate, daunorubicin, doxorubicin, melphalan, cladribine,vincristine, vinblastine, chlorambucil, tamoxifen, taxol, etoposide(VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D,mitomycin C, cisplatin (CDDP), combretastatin(s) and derivatives andprodrugs thereof.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652, for non-limitingexamples of other chemotherapeutic agents that can be used incombination therapies with the CoQ10 compositions. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The physician responsible for administration will be ableto determine the appropriate dose for the individual subject.

Anti-Angiogenics

The term “angiogenesis” refers to the generation of new blood vessels,generally into a tissue or organ. Under normal physiological conditions,humans or animals undergo angiogenesis only in very specific restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonic development and formation of the corpusluteum, endometrium and placenta. Uncontrolled (persistent and/orunregulated) angiogenesis is related to various disease states, andoccurs during tumor growth and metastasis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

As persistent, unregulated angiogenesis occurs during tumor developmentand metastasis, the treatment methods of this invention may be used incombination with any one or more “anti-angiogenic” therapies. Exemplaryanti-angiogenic agents that are useful in connection with combinedtherapy are listed in Table 1. Each of the agents listed therein isexemplary and by no means limiting.

TABLE 1 Inhibitors and Negative Regulators of Angiogenesis SubstancesAngiostatin Endostatin 16 kDa prolactin fragment Laminin peptidesFibronectin peptides Tissue metalloproteinaseinhibitors (TIMP 1, 2, 3,4) Plasminogen activator inhibitors (PAT-1, −2) Tumor necrosis factoralpha (high dose, in vitro) TGF-β1 Interferons (IFN-α, −β, γ) ELR- CXCChemokines:IL-12; SDF-1; MIG; Platelet factor 4 (PF-4); IP-10Thrombospondin (TSP) SPARC 2-Methoxyoestradiol Proliferin-relatedprotein Suramin Thalidomide Cortisone Fumagillin (AGM-1470; TNP-470)Tamoxifen Korean mistletos extract (Viscum album coloratum) RetinoidsCM101 Dexamethasone Leukemia inhibitory factor (LIF)

A certain preferred component for use in inhibiting angiogenesis is aprotein named “angiostatin”. This component is disclosed in U.S. Pat.Nos. 5,776,704; 5,639,725 and 5,733,876, each incorporated herein byreference. Angiostatin is a protein having a molecular weight of betweenabout 38 kD and about 45 kD, as determined by reducing polyacrylamidegel electrophoresis, which contains approximately Kringle regions 1through 4 of a plasminogen molecule. Angiostatin generally has an aminoacid sequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

Certain anti-angiogenic therapies have already been shown to cause tumorregressions, and angiostatin is one such agent. Endostatin, a 20 kDaCOOH-terminal fragment of collagen XVIII, the bacterial polysaccharideCM101, and the antibody LM609 also have angiostatic activity. However,in light of their other properties, they are referred to asanti-vascular therapies or tumor vessel toxins, as they not only inhibitangiogenesis but also initiate the destruction of tumor vessels throughmostly undefined mechanisms. Their combination with the presentinvention is clearly envisioned.

Angiostatin and endostatin have become the focus of intense study, asthey are the first angiogenesis inhibitors that have demonstrated theability to not only inhibit tumor growth but also cause tumorregressions in mice. There are multiple proteases that have been shownto produce angiostatin from plasminogen including elastase, macrophagemetalloelastase (MME), matrilysin (MMP-7), and 92 kDa gelatinase B/typeIV collagenase (MMP-9).

MME can produce angiostatin from plasminogen in tumors andgranulocyte-macrophage colony-stimulating factor (GMCSF) upregulates theexpression of MME by macrophages inducing the production of angiostatin.The role of MME in angiostatin generation is supported by the findingthat MME is in fact expressed in clinical samples of hepatocellularcarcinomas from patients. Another protease thought to be capable ofproducing angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown toproduce angiostatin-like fragments from plasminogen in vitro.

CM101 is a bacterial polysaccharide that has been well characterized inits ability to induce neovascular inflammation in tumors. CM101 binds toand cross-links receptors expressed on dedifferentiated endothelium thatstimulates the activation of the complement system. It also initiates acytokine-driven inflammatory response that selectively targets thetumor. It is an antipathoangiogenic agent that downregulates theexpression VEGF and its receptors.

Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also be used incombination with the present invention. These are both angiogenesisinhibitors that associate with heparin and are found in plateletα-granules. TSP-1 is a large 450 kDa multi-domain glycoprotein that isconstituent of the extracellular matrix. TSP-1 binds to many of theproteoglycan molecules found in the extracellular matrix including,HSPGs, fibronectin, laminin, and different types of collagen. TSP-1inhibits endothelial cell migration and proliferation in vitro andangiogenesis in vivo. TSP-1 can also suppress the malignant phenotypeand tumorigenesis of transformed endothelial cells. The tumor suppressorgene p53 has been shown to directly regulate the expression of TSP-1such that, loss of p53 activity causes a dramatic reduction in TSP-1production and a concomitant increase in tumor initiated angiogenesis.

PF4 is a 70aa protein that is member of the CXC ELR-family of chemokinesthat is able to potently inhibit endothelial cell proliferation in vitroand angiogenesis in vivo. PF4 administered intratumorally or deliveredby an adenoviral vector is able to cause an inhibition of tumor growth.

Interferons and metalloproteinase inhibitors are two other classes ofnaturally occurring angiogenic inhibitors that can be combined with thepresent invention. The anti-endothelial activity of the interferons hasbeen known since the early 1980s, however, the mechanism of inhibitionis still unclear. It is known that they can inhibit endothelial cellmigration and that they do have some anti-angiogenic activity in vivothat is possibly mediated by an ability to inhibit the production ofangiogenic promoters by tumor cells. Vascular tumors in particular aresensitive to interferon, for example, proliferating hemangiomas can betreated with IFNα.

Tissue inhibitors of metalloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix metalloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in combined treatmentprotocols with the present invention. MMPs play a key role in theangiogenic process as they degrade the matrix through which endothelialcells and fibroblasts migrate when extending or remodeling the vascularnetwork. In fact, one member of the MMPs, MMP-2, has been shown toassociate with activated endothelium through the integrin αvβ3presumably for this purpose. If this interaction is disrupted by afragment of MMP-2, then angiogenesis is downregulated and in tumorsgrowth is inhibited.

There are a number of pharmacological agents that inhibit angiogenesis,any one or more of which may be used in combination with the presentinvention. These include AGM-1470/TNP-470, thalidomide, andcarboxyamidotriazole (CAI). Fumagillin was found to be a potentinhibitor of angiogenesis in 1990, and since then the syntheticanalogues of funagillin, AGM-1470 and TNP-470 have been developed. Bothof these drugs inhibit endothelial cell proliferation in vitro andangiogenesis in vivo. TNP-470 has been studied extensively in humanclinical trials with data suggesting that long-term administration isoptimal.

Thalidomide was originally used as a sedative but was found to be apotent teratogen and was discontinued. In 1994 it was found thatthalidomide is an angiogenesis inhibitor. Thalidomide is currently inclinical trials as an anti-cancer agent as well as a treatment ofvascular eye diseases.

CAI is a small molecular weight synthetic inhibitor of angiogenesis thatacts as a calcium channel blocker that prevents actin reorganization,endothelial cell migration and spreading on collagen IV. CAI inhibitsneovascularization at physiological attainable concentrations and iswell tolerated orally by cancer patients. Clinical trials with CAI haveyielded disease stabilization in 49% of cancer patients havingprogressive disease before treatment.

Cortisone in the presence of heparin or heparin fragments was shown toinhibit tumor growth in mice by blocking endothelial cell proliferation.The mechanism involved in the additive inhibitory effect of the steroidand heparin is unclear although it is thought that the heparin mayincrease the uptake of the steroid by endothelial cells. The mixture hasbeen shown to increase the dissolution of the basement membraneunderneath newly formed capillaries and this is also a possibleexplanation for the additive angiostatic effect. Heparin-cortisolconjugates also have potent angiostatic and anti-tumor effects activityin vivo.

Further specific angiogenesis inhibitors, including, but not limited to,Anti-invasive Factor, retinoic acids and paclitaxel (U.S. Pat. No.5,716,981; incorporated herein by reference); AGM-1470 (Ingber et al.,Nature, 48:555-557 1990; incorporated herein by reference); sharkcartilage extract (U.S. Pat. No. 5,618,925; incorporated herein byreference); anionic polyamide or polyarea oligomers (U.S. Pat. No.5,593,664; incorporated herein by reference); oxindole derivatives (U.S.Pat. No. 5,576,330; incorporated herein by reference); estradiolderivatives (U.S. Pat. No. 5,504,074; incorporated herein by reference);and thiazolopyrimidine derivatives (U.S. Pat. No. 5,599,813;incorporated herein by reference) are also contemplated for use asanti-angiogenic compositions for the combined uses of the presentinvention.

Compositions comprising an antagonist of an α_(v)β₃ integrin may also beused to inhibit angiogenesis in combination with the present invention.As disclosed in U.S. Pat. No. 5,766,591 (incorporated herein byreference), RGD-containing polypeptides and salts thereof, includingcyclic polypeptides, are suitable examples of α_(v)β₃ integrinantagonists.

The antibody LM609 against the α_(v)β₃ integrin also induces tumorregressions. Integrin α_(v)β₃ antagonists, such as LM609, induceapoptosis of angiogenic endothelial cells leaving the quiescent bloodvessels unaffected. LM609 or other α_(v)β₃ antagonists may also work byinhibiting the interaction of α_(v)β₃ and MMP-2, a proteolytic enzymethought to play an important role in migration of endothelial cells andfibroblasts.

Apoptosis of the angiogenic endothelium in this case may have a cascadeeffect on the rest of the vascular network. Inhibiting the tumorvascular network from completely responding to the tumor's signal toexpand may, in fact, initiate the partial or full collapse of thenetwork resulting in tumor cell death and loss of tumor volume. It ispossible that endostatin and angiostatin function in a similar fashion.The fact that LM609 does not affect quiescent vessels but is able tocause tumor regressions suggests strongly that not all blood vessels ina tumor need to be targeted for treatment in order to obtain ananti-tumor effect.

Non-targeted angiopoietins, such as angiopoietin-2, may also be used incombination with the present invention. The angiogenic effects ofvarious regulators involve an autocrine loop connected withangiopoietin-2. The use of angiopoietin-2, angiopoietin-1,angiopoietin-3 and angiopoietin-4, is thus contemplated in conjunctionwith the present invention. Other methods of therapeutic interventionbased upon altering signaling through the Tie2 receptor can also be usedin combination herewith, such as using a soluble Tie2 receptor capableof blocking Tie2 activation (Lin et al., Proc. Natl. Acad. Sci., USA,95(15):8829-34, 1998). Delivery of such a construct using recombinantadenoviral gene therapy has been shown to be effective in treatingcancer and reducing metastases (Lin et al., 1998).

CoQ10 Compositions and Combination Therapy with Apoptosis-InducingAgents

The CoQ10 composition treatment may also be combined with treatmentmethods that induce apoptosis in any cells within the tumor, includingtumor cells and tumor vascular endothelial cells. Although manyanti-cancer agents may have, as part of their mechanism of action, anapoptosis-inducing effect, certain agents have been discovered, designedor selected with this as a primary mechanism, as described below.

A number of oncogenes have been described that inhibit apoptosis, orprogrammed cell death. Exemplary oncogenes in this category include, butare not limited to, bcr-abl, bcl-2 (distinct from bcl-1, cyclin D1;GenBank accession numbers M14745, X06487; U.S. Pat. Nos. 5,650,491; and5,539,094; each incorporated herein by reference) and family membersincluding Bcl-xl, Mcl-1, Bak, A1, A20. Overexpression of bcl-2 was firstdiscovered in T cell lymphomas. bcl-2 functions as an oncogene bybinding and inactivating Bax, a protein in the apoptotic pathway.Inhibition of bcl-2 function prevents inactivation of Bax, and allowsthe apoptotic pathway to proceed. Thus, inhibition of this class ofoncogenes, e.g., using antisense nucleotide sequences, is contemplatedfor use in the present invention in aspects wherein enhancement ofapoptosis is desired (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

Many forms of cancer have reports of mutations in tumor suppressorgenes, such as p53. Inactivation of p53 results in a failure to promoteapoptosis. With this failure, cancer cells progress in tumorigenesis,rather than become destined for cell death. Thus, provision of tumorsuppressors is also contemplated for use in the present invention tostimulate cell death. Exemplary tumor suppressors include, but are notlimited to, p53, Retinoblastoma gene (Rb), Wilm's tumor (WT1), baxalpha, interleukin-1β-converting enzyme and family, MEN-1 gene,neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal cancergene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumorsuppressor gene (MTS-1), BRCA1 and BRCA2.

Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469; 5,677,178; and5,756,455; each incorporated herein by reference), Retinoblastoma, BRCA1(U.S. Pat. Nos. 5,750,400; 5,654,155; 5,710,001; 5,756,294; 5,709,999;5,693,473; 5,753,441; 5,622,829; and 5,747,282; each incorporated hereinby reference), MEN-1 (GenBank accession number U93236) and adenovirusE1A (U.S. Pat. No. 5,776,743; incorporated herein by reference) genes.

Other compositions that may be used include genes encoding the tumornecrosis factor related apoptosis inducing ligand termed TRAIL, and theTRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated herein byreference); the 24 kD apoptosis-associated protease of U.S. Pat. No.5,605,826 (incorporated herein by reference); Fas-associated factor 1,FAF1 (U.S. Pat. No. 5,750,653; incorporated herein by reference). Alsocontemplated for use in these aspects of the present invention is theprovision of interleukin-1β-converting enzyme and family members, whichare also reported to stimulate apoptosis.

Compounds such as carbostyril derivatives (U.S. Pat. Nos. 5,672,603; and5,464,833; each incorporated herein by reference); branched apogenicpeptides (U.S. Pat. No. 5,591,717; incorporated herein by reference);phosphotyrosine inhibitors and non-hydrolyzable phosphotyrosine analogs(U.S. Pat. Nos. 5,565,491; and 5,693,627; each incorporated herein byreference); agonists of RXR retinoid receptors (U.S. Pat. No. 5,399,586;incorporated herein by reference); and even antioxidants (U.S. Pat. No.5,571,523; incorporated herein by reference) may also be used. Tyrosinekinase inhibitors, such as genistein, may also be linked to ligands thattarget a cell surface receptor (U.S. Pat. No. 5,587,459; incorporatedherein by reference).

Effective Amounts

The compositions described above are preferably administered to asubject in an effective amount. An effective amount is an amount whichis capable of producing a desirable result in a treated animal or cell(for example, to induce apoptosis or impair mitosis in a cell in theanimal or a culture). As is well known in the medical and veterinaryarts, dosage for any one animal depends on many factors, including theparticular animal's size, body surface area, age, the particularcomposition to be administered, time and route of administration,general health, and other drugs being administered concurrently. It isexpected that an appropriate dosage for topical administration of thecompositions of the invention would be in the range of about 1.5-4.0 mgCoQ10/kg of body weight (e.g., 200 mg for subjects ranging from 110 to300 lbs). An effective amount for use with a cell in culture will alsovary, but can be readily determined empirically (for example, by addingvarying concentrations to the cell and selecting the concentration thatbest produces the desired result). It is expected that an appropriateconcentration would be in the range of about 5-200 μM.

Method for Inhibiting Cancer Cell Growth

The invention provides a method for inhibiting tumor cell growth orincreasing the rate of tumor cell apoptosis. The method includes thesteps of contacting a tumor cell with a composition including asufficient amount of CoQ10 to kill or at least retard mitosis in thetumor cell. The method may be used to inhibit the growth of numeroustypes of cancerous tumor cells. Coenzyme Q10 has been tested and shownto be effective against melanoma, squamous, and breast cancer cells.Coenzyme Q10 is expected to be effective against other cancers as well,particularly those derived from epithelial, mesenchymal, and hemopoieticorigins.

Any suitable formulation of CoQ10 can be used in methods of theinvention. Typical formulations are topical liposomal formulations ofcoenzyme Q10 of varying concentrations. In addition to topicaladministration, CoQ10-containing formulations can be administered to asubject via injection (e.g., IP, IV, IM, SQ).

In a method of reducing the rate of tumor cell growth or increasing therate of tumor cell apoptosis in vitro, CoQ10 is dissolved in 2-propanolfollowed by dilution in a desired medium (as described in example 1below). In an in vivo method of reducing the rate of tumor cell growthor increasing the rate of tumor cell apoptosis, a CoQ10-containing creamis applied topically daily to the target site until tumor regressionoccurs (as described in examples 2 and 3). In another in vivo method, aCoQ10-containing formulation is administered to a subject via injection(e.g., IP, IV, IM, SQ).

Inhibition of tumor cell growth manifested by administration of theCoQ10 compositions described herein, that is compositions comprisingabout 1% to about 25% coenzyme Q10, refers to one or more of thefollowing effects: (1) inhibition, to some extent, of tumor growth,including, (i) slowing down and (ii) complete growth arrest; (2)reduction in the number of tumor cells; (3) maintaining tumor size; (4)reduction in tumor size; (5) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of tumor cell infiltrationinto peripheral organs; (6) inhibition, including (i) reduction, (ii)slowing down or (iii) complete prevention, of metastasis; (7)enhancement of anti-tumor immune response, which may result in (i)maintaining tumor size, (ii) reducing tumor size, (iii) slowing thegrowth of a tumor, (iv) reducing, slowing or preventing invasion and/or(8) relief, to some extent, of the severity or number of one or moresymptoms associated with the disorder.

In preferred embodiments, administration of coenzyme Q10 compositionsresults in one or more phenotypes of a tumor cell being inhibited. Forexample, inhibition of tumor growth, reduction of tumor size, inhibitionof metastasis, reduction in the number of tumor cells and the like. Eachof these phenotypes of a tumor cell can be measured using standardassays, such as for example, imaging, mechanical measurements, in vitroassays and the like.

Kit and Formulations

The invention also provides a kit for reducing the rate of tumor growthin a subject. The kit of the invention includes a composition comprisingCoQ10 and a pharmaceutically acceptable carrier as well as printedinstructions for using the composition to reduce the rate of tumorgrowth in a subject.

Active components can be present in solid, semi-solid or liquid form.Solid forms include for example, powders, granules and flakes.Semi-solid forms include, for example, gels, creams, gelatins andointments. These and other active agents embraced by the presentinvention are known to those of ordinary skill in the art and, in mostcases, are commercially available from suppliers such as CompoundSolutions, Inc., Escondido, Calif. Information on these and other activeand inactive agents embraced by the invention, and their commercialsuppliers is available from various trade manuals, most particularly,Remington's Pharmaceutical Sciences, United States Pharmacopoeia (USP),National Formulary (NF), Merck Index, Physician's Desk Reference (PDR)and Chemical Abstracts.

The kits of the invention will also generally contain at least oneinactive agent. As used herein, inactive agents are agents which do notprovide any therapeutic benefit to the subject to whom they areadministered. Instead, inactive agents can function in many other wayssuch as to provide a base in which the active agent can be dissolved orsuspended, to dilute the active agent in order to provide proper dosesupon administration, to facilitate the dissolution or suspension of theactive agent, or to prevent oxidation of the active agent by removingair bubbles from the final compounded suspension. In some embodiments ofthe invention, the kits lack an inactive agent, and rather contain twoor more active agents.

Base agents such as creams, oils, gels or ointments are suitable fortopical or suppository applications. The choice of suitable inactivebase agent for use in the kits of the invention will depend upon theactive agent to be compounded. Suitable base agents will be known to theordinary artisan, Alternatively, Remington's Phamtaceutical Sciences,the Physician Desk Reference (PDR) or other manuals as listed above, canbe consulted in making this determination.

Examples of inactive base agents or components include, for example,lanolin, hydrophilic ointment, white ointment, yellow ointment,polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, whitepetrolatum, rose water ointment, squalene, hydrogenated vegetable oil(Type II), ultrasound gel, pluronic lecithin organogel (PLO) gel, cream.

The term “petrolatum” as used herein means petrolatum ointment,petrolatum gel or petrolatum cream, all of which are commerciallyavailable. It is well within the realm of the ordinary pharmaceuticalartisan to determine which form of petrolatum is most appropriate for aspecific kit.

A commercially available ultrasound base is either POLYSONIC™(ultrasound gel) ultrasound lotion or Aquasonic ultrasound 100 gelmanufactured by Parker Laboratories, Inc. (Fairfield, N.J.) or EcoGel100 or EcoGel 200 manufactured by Eco-Med (Mississauga, Ontario,Canada), the compositions of which may include cetyl alcohol, liquidparaffin, polymer, surfactants, preservatives such as propyl paraben andmethyl paraben in bacteriostatic concentration, fragrance, and reverseosmosis water. As used herein, a gel is a base with a higher viscositythan a lotion. The physical characteristics of the POLYSONIC™(ultrasound gel) ultrasound lotion and the EcoGel 100 include pH rangeof 6.5-7.0, density of 1.04 g/cm³, viscosity of 35,000 to 70,000 cps andacoustic impedance of 1.60 (10⁵ g/cm² sec). The physical characteristicsof Aquasonic ultrasound 100 gel or EcoGel 200 are similar to those ofPOLYSONIC™ (ultrasound gel) ultrasound lotion and EcoGel 100 except thattheir viscosity is 80,000 to 110,000 cps. These lotions and gels areavailable in a clear, colorless form or in a blue colored form.

Liquid bases are recommended for orally administered pharmaceuticals. Insome embodiments of the invention, at least one active agent, e.g.CoQ10, will be supplied already co-mingled with an inactive agent.Examples of this include the combination of magnesium hydroxide andaluminum hydroxide (commercially available as MAALOX™ (magnesiumhydroxide/aluminum hydroxide)), and diphenhydramine HCl (commerciallyavailable as BENADRYL™ (diphenhydramine hydrochloride)). Both MAALOX™(magnesium hydroxide/aluminum hydroxide) and BENADRYL™ (diphenhydraminehydrochloride) are supplied by their respective manufacturers as acombination of active and inactive agents.

Sterile base solutions are preferred for parenteral (i.e., injection),aerosol (i.e., inhalation) and ophthalmic routes of administration. Theadministration may, for example, be intravenous, intraperitoneal,intramuscular, intracavity, subcutaneous or transdermal. Preparationsfor parenteral administration includes sterile aqueous or nonaqueoussolutions, suspensions and emulsions. The compounded pharmaceuticals,preferably those intended for parenteral, inhalation or ophthalmicroutes of administration, may be prepared and administered in inactiveagents which are pharmaceutically-acceptable. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive agents and that is compatible with the biological systems such ofa tissue or organism. The physiologically acceptable carrier must besterile for in vivo administration. Pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers andother materials which are well-known in the art. The characteristics ofthe carrier will depend on the route of administration. In general,pharmaceutically-acceptable agents or carriers are well-known to thoseof ordinary skill in the art. In some embodiments, suitable sterilesolutions include albuterol and ipratropium inhalation solution;papaverine, phentolamine and prostaglandin injection solution; fentanylcitrate injection solution and cyclosporine ophthalmic drops.

Examples of nonaqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, an injectable organic esterssuch as ethyloliate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, (such as those based on Ringer's dextrose),and the like. Preservatives and other additives may also be present suchas, for example, antimicrobials, antioxidants, chelating agents, andinert gases and the like. Those of skill in the art can readilydetermine the various parameters for preparing these alternativepharmaceutical compositions without resort to undue experimentation.

Inactive agents may also include components which function to preservethe integrity of the compounded formulation. This latter category ofinactive agents includes, for example, anti-foaming agents. Anti-foamingagents are agents which function to remove unwanted air trapped in acomposition, perhaps during mixing or agitation. The use of anti-foamingcomponents is particularly useful in the preparation of pharmaceuticalsto be used for ultrasound imaging due to the impedance of signaltransmission by air bubbles. Examples of other anti-foaming agentsuseful in the compositions of the invention includebisphenylhexamethicone, dimethicone, dimethiconol, hexamethyldisiloxane,hexyl alcohol, isopropyl alcohol, petroleum distillates, phenethyldisiloxane, phenyl trimethicone, polysilicone-7, propyl alcohol, silicadimethyl silylate, silica silylate, tetramethyl decynediol andtrimethylsiloxysilicate. A preferred anti-foaming agent is simethicone.Simethicone is a mixture of about 90% dimethicone and 10% siliconedioxide (w/w). Simethicone is used extensively as an anti-gas agent inpharmaceutical products such as GAS-X™ (simethicone), MAALOX™ (magnesiumhydroxide/aluminum hydroxide), MYLANTA™ (aluminum, magnesiumsimethicone), PHAZYME™ (simethicone), GENAZYME™ (simethicone), andMYLICON™ (simethicone) Drops. Simethicone may be used as on anti-foamingagent in any of the formulations embraced by the invention.

Other inactive agents which can be included in the formulations of theinvention include stabilizers such as citric acid, anti-oxidants such assodium metabisulfite and preservatives such as methyl or propyl paraben.

Another class of inactive agents is suspending agents. Suspending agentsare agents which facilitate the suspension and in some cases thedissolution of an active agent in a base. Generally, suspending agentsensure more uniform mixing of active and base components. In order toadminister a more uniform dose of a compounded pharmaceutical to apatient, the compounded components must be properly and homogeneouslycombined. If the active agent is present as a powder, a uniformdispersion is sometimes difficult to achieve using the traditional formof compounding.

A subcategory of suspending agents are solubilizers. Solubilizers areagents which facilitate the dissolution of a solid or, in some cases, asemi-solid agent in a base inactive agent. In some embodiments of theinvention, a solid-form active agent may be dissolved in a suspendingagent, prior to mixing it with the base agent. Conversely, thesuspending agent and the base agent may be prepackaged together,particularly if the concern is ensuring the uniform blending of activeagent within the base component rather than the loss of solid (i.e.,powdery) active agent. In still other variations, the suspending agentmay be premixed with the base inactive agent.

Suitable suspending agents useful in the compositions of the inventioninclude, but are not limited to, glycerin, hexylene glycol, propyleneglycol, sorbitol, acacia, cholesterol, diethanolamine (adjunct),glyceryl monostearate, lanolin alcohols, lecithin, mono- anddi-glycerides, monoethanolamine (adjunct), oleic acid (adjunct), oleylalcohol (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleylether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate, sorbitan monostearate, stearic acid, trolamine,emulsifying wax, benzalkonium chloride, benzethonium chloride,cetylpyridinium chloride, docusate sodium, nonoxynol 9, nonoxynol 10,octoxynol 9, polyoxyl 50 stearate, and tyloxapol.

Still other suspending agents include humectants and wetting agents.Humectants are agents which retain moisture. Examples of humectantsinclude but are not limited to glycerin, hexylene glycol, propyleneglycol and sorbitol. The amounts of base and non-base inactive agentswill also depend upon the particular compounded pharmaceutical to bemade. Base agents can be provided in quantities corresponding to finalcompounded preparations which contain 0.5% to 99.99% of base agent,either in weight or in volume. In preferred embodiments, the finalconcentration of the base agent is 20%-80%. In even more preferredembodiments, the final concentration of the base agent is 40%-80%.

Generally, the amounts of non-base agents will be sufficient to providefinal formulations in which each non-base inactive agent represents0.01%-50% (w/w) of the composition. Suspending agents may represent1%-50% (w/w) of the final formulation. Preferably, suspending agentswill represent 1%-40% and even more preferably, they will represent5%-30% of the final formulation. Anti-foaming agents may represent 0.01%to 20% (w/w) of the final formulation. More preferably, anti-foamingagents represent 0.05% to 10% of the final formulation and even morepreferably, they represent 0.1% to 5% of the final formulation.

In some preferred embodiments, the single or multiple unit of use kitsare designed to yield, after the physical mixing of active and inactiveagents, compounded pharmaceutical formulations comprising 1%, 5%, 10% or20% w/w of CoQ10.

The kits of the invention will provide each and every component requiredfor preparing a given compounded pharmaceutical in pro-measuredquantities. The measuring of each component will be performed usingcurrent Good Manufacturing Practices (cGMP, as legislated by the Code ofFederal Regulations or CPR), as will the packaging and labeling of eachcomponent and the final packaging and labeling of the kit in itsentirety. In this way, the kits are standardized and variations frombatch to batch will be minimal or non-existent and the precision andaccuracy in the measurement of individual components will be improvedconsiderably over the methods currently used by pharmacists.Instructions may be provided as separate from any container, but stillcontained in the kit. Alternatively, instructions mnay be located on acontainer, for example, on an exterior surface or on an interior surfacesuch as a lid.

Both the active and the inactive agents of the kit are provided incontainers. Since the kit will contain at least one active and at leastone inactive agent, or at least two active agents pre-formulated withinactive agents, the minimum number of containers in a given kit will betwo. In preferred embodiments, the maximum number of containers in a kitwill be less than or equal to four. The containers may be formed in anysize or shape useful for the mixing or transferring of components fromone container to another. For example, each container may be in the formof vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes orpouches, tubes or blister packages or any other suitable form providedthe container is sealed so as to prevent premature mixing of components.As used herein, a container may also be a compartment or a chamberwithin a vial, a tube, a jar, or an envelope, or a sleeve, or a blisterpackage or a bottle, provided that the contents of one compartment arenot able to associate physically with the contents of anothercompartment prior to their deliberate mixing by a pharmacist orphysician.

The invention intends to provide within a single kit all the necessarycomponents, containers and stirring or mixing elements for preparing aunit of use compounded pharmaceutical without the need for otheraccessories. The kits of the invention may also contain items such asgloves or spill pads. Individuals skilled in the art can readily modifythe choice of container to suit the individual components housed andmixed therein.

In some embodiments of the invention, the final compounded formulationwill be provided to the patient in the container originally housing theinactive, or base, compound. In other embodiments, the final compoundedformulation will be provided in the container originally housing theactive agent. In still other embodiments, all the necessary componentsfor preparing a compounded pharmaceutical are included in one containerbut are physically separated within such a container. For example, aninactive agent may be contained in the lower part of a container, suchas a jar, and may be covered by a plastic, peel-off wrap. The activeagent may be housed in this same jar, but secured to the lid of the jarand provided in a pouch or a sleeve. The ability to provide allcomponents together in the smallest packaging arrangement may bepreferable in some circumstances. Mixing elements required in thepreparation of the compounded pharmaceutical may also be located withinthe same container, for example, secured to the inside surface of thelid of the container.

In still another embodiment of the invention, active and inactive agentsare provided in adjacent compartments of a single housing container, andare mechanically removed from these compartments and into a thirdcompartment. As an example, all the chemical components necessary toprepare a particular compounded pharmaceutical can be present in asingle tube, for example, a tube similar to a toothpaste tube having aninterior which is divided into separate compartments. Bach of thesecompartments in turn house a base agent or an active agent. Either thebase agent or the active agent may be premixed with an anti-foamingagent and/or a suspending agent, as described herein. By applyingpressure on the tube as a whole, the components are made to exit theirrespective compartments. They can then be mixed either in an adjacent ora physically separate compartment. Squeezing or pressing of the outsidesurface of the tube may be all that is necessary to retrieve theindividual components housed within the tube. In yet another embodiment,the contents of both chambers of a container can be pumped out and intoa third container. In a related embodiment, it is also envisioned thatrather than requiring the contents of each compartment to exit and flowinto a third compartment, the components may be separated by a removablesheet or film. Thus, upon removal of such a sheet or film, the contentsof the two compartments are in contact and may require only agitation orend-over-end inversion to become completely mixed. This latterembodiment would eliminate the need for a mixing element, andpotentially for an exterior package particularly if the instructions arewritten on the container itself.

According to some aspects of the invention, each container may containone or more active agents or one or more inactive agents. For example,in some embodiments of the invention, none of the containers may containboth an active and an inactive agent prior to mixing by the pharmacistor physician. However, the invention also provides for kits in which acontainer may contain an active and at least one inactive agent, such asa base agent, a suspending agent or an anti-foaming agent.

In a preferred embodiment, the active agent is provided premixed with aninactive agent. This applies mainly when CoQ10 is commercially availableas a solid, for example a powder, and the pre-mixing of the powder witha suspending agent facilitates the compounding by the pharmacist orphysician. In yet other embodiments, at least two of the inactive agentsmay be pre-mixed as provided in the kits of the invention.

In some embodiments, where the active agent is added to the basecomponent, it may be desirable to provide the base component in acontainer which is only partially full. In preferred embodiments, thecontainer in which the base component is situated is less than 100% fullby volume. In other embodiments, the containers are 95%, 90%, 80%, 75%,70%, 60%, 50%, 40%, 30%, 25%, 20% or less than 20% full by volume. Inother embodiments, the active or inactive agents comprise a volume oftheir respective containers ranging from 100% to greater than 1%, andevery integer there between. In preferred embodiments, the inactiveagent occupies a volume of the second container which is less than orequal to the volume of the second container minus the volume of theactive agent.

As used according to the invention, the active and inactive agents arephysically combined by a pharmacist to produce a compoundedpharmaceutical. The components of the kit can be combined by gentleagitation, shaking, stirring, folding or end-over-end inversion of thefirst or second container. In some instances, the proper mixing of theactive and inactive agents may be accomplished simply by adding one tothe other, followed by scaling and agitation of the container. This isespecially the case if the components are both liquids or bothsemi-solids. In other instances, it may be necessary to stir thecomponents together with a mixing element. Mixing elements are wellknown to a person of ordinary skill in the pharmaceutical arts and mayinclude for example, centrifuges, a mixing rod such as a glass rod, aspoon, a spatula or a dipstick. Where required, the mixing element isprovided in the kit. The presence of a mixing element will varydepending on the compounded pharmaceutical formulation to be made withthe components of a kit.

The final compounded pharmaceutical may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, suppositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces locally administering thecompounded pharmaceuticals of the invention such as, for example, asimplants. These formulations may be intended for oral, topical, mucosal,parenteral (e.g., injectable), rectal or vaginal administration. Inpreferred embodiments, the final compounded formulations may beself-administered.

The kits of the invention may also contain a package which may becompartmentalized to receive in close confinement two or more containersof the invention. In some embodiments, the package may be box-like,being made of a moderately rigid material such as cardboard orreinforced paper. In other embodiments, the package may be a bag. Instill other embodiments, as described herein, there is no externalpackaging and all containers may be incorporated into one of thecontainers housing either an active or an inactive agent. This latterembodiment can be accomplished by securing containers such as pouches,sleeves or sacs, containing either active or inactive agents, as well asany mixing elements required for the compounding, to the interior of thelid of the main container. An individual skilled in the art can readilymodify the package to suit the individual needs of each kit and eachuse. The kits of the invention further contain instructions for theproper use of the components found therein.

The kits of the invention are intended for use in the treatment orprevention of a number of disorders in a variety of subjects includinghumans, dogs, cats, horses, fish, pigs, cows, sheep, deer, zoo animalsand laboratory animals (e.g., mice, rats, rabbits, monkeys, etc.). Theinvention intends to embrace unit of use kits containing the abovepreparations.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

All publications and patent documents cited in this application areincorporated by reference in pertinent part for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

EXAMPLES

The following examples serve to illustrate the invention withoutlimiting it thereby. It will be understood that variations andmodifications can be made without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods for Apoptosis Assay

Cell lines used in the assay were SK-Mel28 and nFIB. Cells (SK-Mel28 andnFIB) were seeded (5×10⁴ cells/well) into wells containing either solelymedium or medium with treatment and placed in an incubator at 37° C., 5%CO₂, and under humidified conditions for 48 hours. Each condition wasperformed in duplicate and was subjected to the following protocol:

Apoptosis Analysis as Per Protocol of BD Pharmingen Annexin-VPE Protocol

Reagents include Annexin V-PE (BD Pharmingen, San Diego, Calif.), 7-AAD(BD Pharmingen, San Diego, Calif.), binding buffer (10×: 0.1 MHepes/NaOH, 1.4 M NaCl, 25 mM CaCl₂) [diluted to 1× (9 mL PBS and 1 mLbinding buffer) for use in experiment] (BD Pharmingen, San Diego,Calif.), Trypsin-EDTA (Gibco, Grand Island, N.Y.), and desired Media.

Add 0.5 mL trypsin to each well, remove trypsin after approximately 10seconds, and add 0.5 mL trypsin to each well. Place wells in anincubator, observe level of detachment under microscope after 4 minutes,and gently tap sides and bottom to aid in detachment. When cells detach,neutralize with 0.5 mL serum-supplemented medium. Transfer cell solutionto centrifuge tubes, centrifuge cells at 2000 RPM for 5 minutes,aspirate supernatant, resuspend in 6 mL PBS, and split 6 mL into threecentrifuge tubes (2 mL each). Centrifuge cells at 2000 RPM for 5minutes, aspirate supernatant, resuspend in 100 μL binding buffer mix,add 50 μL of Annexin V-PE and 50 μL of 7-AAD in each centrifuge tube,and vortex and place in the dark for 15 minutes. Add 350 μL bindingbuffer to each tube and perform analysis using the flow cytometer.

A baseline was also created using freshly cultured cells from a flask.The cells were subcultured and washed twice with cold PBS. Subsequently,they were resuspended in 1× binding buffer to a concentration of 1×10⁶cells/mL. 100 μL of cell suspension were transferred into three testtubes for a total of 1×10⁵ per tube. One tube served as a negativecontrol with no staining introduced. Another was stained with onlyAnnexin V-PE while the final was stained with only 7-AAD. 50 μL ofstaining solution was placed into each of the tubes. These tubes werethen placed in the dark for 15 minutes after which time, 350 μL ofbinding buffer were added to each. They were then subjected to analysisby flow cytometry prior to the treated and control cells.

Experiment 1: The Effect of Coenzyme Q10 on the level of Apoptosis inHuman Breast Cancer Cells MCF-7 MCF-7 MCF-7 Control Control Control 100μM 100 μM 100 μM CoQ10 CoQ10 CoQ10 Seeded 50,000 cells/well Compare toBaseline 100,000 cells/sample in Apoptosis Assay (Annexin PI) after 72hrs

Experiment 2: The Effect of 2-Propanol Vehicle on the level of Apoptosisin Melanoma Cells SK-MEL 28 SK-MEL 28 SK-MEL 28 Control Control ControlEquivalent Vol. if Equivalent Vol. if Equivalent Vol. if 50 μM of CoQ1050 μM of CoQ10 50 μM of CoQ10 (1% 2-Propanol) (1% 2-Propanol) (1%2-Propanol) Seeded 50,000 cells/well Compare to Baseline 100,000cells/sample in Apoptosis Assay (Annexin PI) after 48 hrs

Experiment 3: The Effect of 2-Propanol Vehicle on the level of Apoptosisin Neonatal Fibroblasts nFIB (P) 6 nFIB (P) 6 nFIB (P) 6 Control ControlControl Equivalent Vol. if Equivalent Vol. if Equivalent Vol. if 50 μMof CoQ10 50 μM of CoQ10 50 μM of CoQ10 (1% 2-Propanol) (1% 2-Propanol)(1% 2-Propanol) Seeded 50,000 cells/well Compare to Baseline 100,000cells/sample in Apoptosis Assay (Annexin PI) after 48 hrsPreparation of DMEM/F12 MediumMaterials:

DMEM/F12 medium (Cat#11330-032 Gibco-Invitrogen Corp, Grand Island,N.Y.)

Siliconized Sterile Pipette tips—1 mL and 25 mL to be used withPipettMan

FBS (Fetal Bovine Serum) Supplement (Gibco-Invitrogen Corp, GrandIsland, N.Y.)

PSA (Penicillin Streptomycin Amphoterocin B)—Antimicrobrial AgentSupplement (Cascade Biologics, Inc., Portland, Oreg.)

Procedures:

Transfer appropriate amount of FBS into DMEM/F12 (e.g., 50 mL FBS in 500mL medium for 10% serum concentration). Add appropriate amount of PSA toobtain a solution with a final concentration of 100 U/mL Penicillin G,100 μg/mL streptomycin sulfate, and 0.25 μg/mL Amphotericin B (e.g., 1mL of 500×PSA in 500 mL medium). Mix by pipetting and inverting bottle.Store at 4° C. until use.

Preparation of EpiLife Medium

Materials:

Siliconized Sterile Pipette tips—5 mL, 10 mL to be used with PipettMan

EpiLife Media (M-EPI-500, Cascade Biologicals)

PSA (500× Penicillin Streptomycin Amphoterocin B)—Antimicrobrial AgentSupplement (R-004-10 Cascade Biologics)

EDGS (Epidermal Growth Supplement) (S-012-5 Cascade Biologics)

Procedures:

Transfer one vial of EDGS (5 mL) and PSA (1 mL) into EpiLife Mediumresulting in 100 U/mL Penicillin G, 100 μg/mL streptomycin sulfate, and0.25 μg/mL Amphotericin B (e.g. 1 ml, of 500×PSA in 500 mL medium). Mixby pipetting and inverting. Store in 4° C. until use.

Creating a Homogenous Solution of Q10 in Media Protocol

Materials:

Polystyrene Sterile Pipette tips—200-1000 μM to be used with automaticpipettes

Siliconized Sterile Pipette tips—10 mL to be used with PipettMan

15 mL Centrifuge Tubes

Media

Coenzyme Q10 (Compound Solutions, Inc., Escondido, Calif.)

2-propanol (Cat#9083-3, J.T. Baker Chemical Co., Phillipsbury, N.J.)

Procedures:

Retrieve Q10 stock from −20° C. storage and weigh out approximately 4.4mg. Transfer Q10 into a 25 mL centrifuge tube. Add 1 mL 2-propanol tocentrifuge tube. Vortex and dip in hot water bath (55° C.) to promotedissolution. Add 9 mL of media to centrifuge tube. Vortex and dip in hotwater bath (55° C.) if necessary to create a homogenous solution. Thisresults in a 500 μM Q10 solution, Make serial dilutions to treatmentconcentrations.

Defrosting Cells Protocol

Materials:

Siliconized Sterile Pipette tips—1 mL, 10 mL to be used with PipettMan

75 cm² Cell Culture Flasks

15 mL Centrifuge Tubes

Procedures:

Acclimate reagents to 37° C. in water bath. Remove cells from liquidnitrogen tank. Keep vial clasped in palm to initiate defrost. Submergein water bath at 37° C. until completely melted. Transfer cells to a 15mL centrifuge tube with 10 mL of growth medium. Mix by pipetting.Centrifuge at 2500 RPM for 8 minutes. Aspirate supernatant. Resuspendpellet with appropriate medium. Mix by vortexing and pipetting tohomogenize cell suspension. Transfer to 75 cm² Cell Culture flask(s).

Subculturing Cells Protocol

Materials:

Siliconized Sterile Pipette tips—5 mL, 10 mL to be used with PipetteMan

75 cm2 (T75) Cell Culture Flasks

6 Well Tissue Culture Plates

15 mL Centrifuge Tubes

Media

0.05% Trypsin (Cat#25-052-C1-1X Trypsin-EDTA, Cellgro by Mediatech,Herndon, Va.)

Procedures:

Acclimate reagents to 37° C. in water bath. Remove medium from cellculture flasks (cells are ready for subculture when approximately 85%confluent). Prime by adding 1-2 mL of trypsin to flask for 30 seconds.Remove trypsin from flask. Add 5 mL of trypsin to flask. Place flask inthe incubator at 37° C. for approximately 4 minutes. Remove and observedegree of detachment with microscope. If needed, gently tap flask to aidin detachment. Add 5 mL of serum-containing medium. Mix by pipetting andwashing flask with cell suspension. Transfer cell suspension to a 15 mLcentrifuge tube. Vortex centrifuge tube. Centrifuge at 2500 RPM for 8minutes. Aspirate supernatant. Resuspend pellet in appropriate medium.Create a homogenous cell suspension by pipetting and vortexing. Seedcells in new T75 flasks or into wellplates for experimentation.

Counting Cells Protocol

Materials:

Beckman Coulter® Z1 Cell and Particle Counter (Beckman Coulter, Inc.,Fullerton, Calif.)

Coulter Counter Vials (Beckman Coulter, Inc.)

Isoton II Diluent (#8546719, Beckman Coulter)

Coulter CLENZ (#8546929, Beckman Coulter)

Polystyrene Sterile Pipette tips—20-200 μM, 200-1000 μM to be used withautomatic pipettes

Procedures:

After subculture (per subculturing cells protocol described above),pipet the desired volume suspension of cells to count (0.25-1 mL) intoCoulter Counter Vial (Beckman, Inc.) using an automatic pipette. Insurethat the Beckman Coulter® Z1 Cell and Particle Counter is clean by usingCoulter CLENZ (Beckman, Inc., Fullerton, Calif.) to flush. Flushapparatus once with Isoton II Diluent. Add Isoton II Diluent to vialcontaining cells for a total volume of 10 mL. Use output mode ofapparatus to count cells twice to ensure accuracy. Average countstogether and calculate total cell number per volume.

Performing In Vitro Experiments Protocol

Materials:

Polystyrene Sterile Pipette tips—20-200 μM, 200-1000 μM to be used withautomatic pipettes

Siliconized Sterile Pipette tips—5 mL, 10 mL to be used with PipettMan

75 cm2 Cell Culture Flasks

6 Well Tissue Culture Plates

15 mL Centrifuge Tubes

Coulter Counter Vials (Beckman Coulter, Inc.)

0.05% Trypsin (Cat#25-052-C1-1X Trypsin-EDTA, Cellgro)

Procedures:

Acclimate reagents to 37° C. in water bath. Make stock solution of Q10as per protocol described above for creating a homogenous solution ofQ10 in media. Perform serial dilutions to desired concentrations. Place2 mL media into respective wells. Subculture flasks as per protocoldescribed above for subculturing cells. Resuspend cells with just enoughmedium to create a homogenous cell suspension (approximately 5 mL).Determine cell concentration as per protocol described above forcounting cells. Dilute cell suspension so that the desired amount ofcells to seed is contained within 50-100 μL. Seed desired amount ofcells into each well. Incubate at 37° C., 5% CO₂, and under humidifiedconditions for desired duration. Aspirate media from wells. Place 0.5 mLtrypsin into each well. Incubate for approximately 4 minutes. Check fordegree of detachment under microscope. Swirl, gently tap sides, andgently knock bottom to aid in detachment if necessary. Neutralizetrypsin with 0.5 mL medium. Pipette to aid in cell detachment andbreaking of clumps. Remove 0.5 mL cell suspension and place in coultercounter vials (Beckman Coulter, Inc.). Count cells as per protocoldescribed above for counting cells.

Inoculation of Animals Protocol

Materials:

Phosphate buffer solution (PBS) (Gibco-Invitrogen Corp, Grand Island,N.Y.)

Polystyrene Sterile Pipette tips—20-200 μM, 200-1000 μM to be used withautomatic pipettes

Siliconized Sterile Pipette tips—5 mL, 10 mL to be used with PipettMan

75 cm² Cell Culture Flasks

15 mL Centrifuge Tubes

Coulter Counter Vials (Beckman Coulter Inc.)

0.05% trypsin (Cat#25-052-C1-1X Trypsin-EDTA, Cellgro)

Centrifuge tubes (2 mL)

Anesthetic (Aventin)

Procedures:

Subculture flasks as per the cell subculturing protocol described above.After aspirating supernatant, combine pellets from each flask dilutedslightly with PBS with a 5 mL pipette. Dilute final cell suspension tocontain approximately ten million cells per 100 μL. Transfer cellsuspension to micro-centrifuge tubes (2 mL). Place in ice immediatelyand leave in ice until injected. Anesthetize mice via an intraperitonealinjection with 0.3 cc Aventin. Inoculate each animal subcutaneously with0.1 cc cell suspension per site. Transfer any remaining cells into a 15mL centrifuge tube, Dilute to 10 mL with medium. Centrifuge at 2500 RPMfor 8 minutes. Aspirate supernatant. Add 10 mL media to centrifuge tube.Create a homogenous cell suspension by pipetting and vortexing. Seedcells in a T75 flask to ensure experimental cell viability.

Example 2 Effect of a Topical Formulation of Coenzyme Q10 on SK-MEL28Tumors in Mice

Melanoma tumors were induced in mice by SK-MEL28 injection into thesubcutaneous layer. The animal study consisted of both a control andtreatment group each containing four mice. The mice were inoculated withtwo tumors and the graph of FIG. 14 represents the resulting mean massfor the tumors in each mouse. A topical formulation of Coenzyme Q10 (A1.0% and 1.5% formulation was tested) was applied to the tumors in thetreatment group daily for a period of 30 days. After which, the tumorswere excised and the mass was determined. The difference in the overallmean mass of the treatment group was significant compared to the control(P<0.05).

Example 3 Preparation of Topical CoQ10 Cream

Reagents:

-   -   Phospholipon 90G (American Lechitin, Stanford, Conn.)    -   Glycerol    -   BHT    -   Ethanol    -   MCT    -   lavender (Sigma-Aldrich)    -   CoQ10 (Pure Prescriptions, San Diego, Calif.)        Procedure:

10 g of Phospholipon 90G (American Lechitin, Stanford, CT) and 5 g ofPhospholipon 90H were dissolved in a mixture of 13.3 g of Glycerol(Sigma-Aldrich, St. Louis, MO), 0.3 g BHT (Sigma-Aldrich), 9 ml ethanol(Sigma-Aldrich), and 1.5 g MCT (Sigma-Aldrich) at 60° C. 1.1 g of CoQ10(Pure Prescriptions) were dissolved into the resulting mixture. 65 ml of1 mM phosphate buffer (pH 8.2) prepared with nitrogen saturated waterand 0.2 ml of lavender (Sigma-Aldrich) were added and the mixture wasblended in a high speed blender at 12,000 RPM to form a cream. The creamwas stored at 4° C. until used.

Example 4 Apoptosis Analysis for JC-1 Stain

Apoptosis was measured using a mitochondrial membrane dye JC-1,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyaninechloride (Molecular Probe, Eugene, Oreg.). Treatments consisting ofDMEM-F12 media supplemented with 1×PSA, 5% FBS and 0, 50, 100, and 200μM concentrations of coenzyme Q10 were prepared in 60×15 mm tissueculture dishes (Costar—Cambride, Mass.). PC-3 cells were seeded at500,000 cells per dish and incubated for 24 hours. The cells weretrypsinized using 2 mL trypsin-EDTA and subjected to centrifugation at2,500 rpm for 8 minutes. They were resuspended in 1 mL of Ham's F12medium lacking serum and phenol red (Cascade Biologics, Inc—Portland,Oreg.) and promptly placed on ice. A 1 mg/ml stock solution of JC-1 wasprepared using sterile DMSO and 10 μL was added to each cell suspensionwhile gently vortexing. The cells were incubated at 37° C. for 15 min,diluted with 4 ml of Ham's F12 medium and centrifuged at 600 rpm for 7min. Resuspended in 5 ml of cold PBS (Gibco—Grand Island, N.Y.), thecells were centrifuged again at 600 rpm for 7 min. The cell pellet wasthen suspended in 1 ml of cold PBS and transferred to nylon filter topflow cytometry tubes covered with foil to prevent light penetration. Thesamples were analyzed by flow cytometry for changes in uptake offluorescent dye. The monomer JC-1 displays green fluorescence(λ_(em)=527 nm) while the J-aggregates display red fluorescence(λ_(em)=590 nm). Permeabilized mitochondria accumulate the JC-1 monomerdye prior to and during apoptosis.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspect, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating a pancreatic tumor in ahuman subject comprising intravenously administering to the subject acomposition comprising 1% to 20% w/w of Coenzyme Q10 in an amounteffective and for a sufficient time to slow the growth of the pancreatictumor, thereby treating the pancreatic tumor in the subject.
 2. Themethod of claim 1, wherein the composition comprises 1% to 10% w/w ofCoenzyme Q10.
 3. The method of claim 1, wherein the compositioncomprises 1% to 5% w/w of Coenzyme Q10.
 4. The method of claim 1,wherein the Coenzyme Q10 composition is administered with an additionalanti-cancer agent.
 5. The method of claim 1, comprising intravenouslyadministering Coenzyme Q10 by continuous infusion.
 6. The method ofclaim 4, wherein the additional anti-cancer agent is co-administeredwith the composition comprising Coenzyme Q10 to the subject.
 7. Themethod of claim 4, wherein administration of the additional anti-canceragent precedes administration of the composition comprising Coenzyme Q10to the subject.
 8. The method of claim 4, wherein administration of theadditional anti-cancer agent follows administration of the compositioncomprising Coenzyme Q10 to the subject.
 9. The method of claim 4,wherein the additional anti-cancer agent is an anti-angiogenic agent.10. The method of claim 4, wherein the additional anti-cancer agent is achemotherapeutic agent.
 11. The method of claim 10, wherein thechemotherapeutic agent is selected from the group consisting ofcyclophosphamide, taxanes, busulfan, methotrexate, daunorubicin,doxorubicin, melphalan, cladribine, vincristine, vinblastine,chlorambucil, tamoxifen, taxol, camptothecin, actinomycin-D, mitomycinC, combretastatin, cisplatin, etoposide, verapamil, podophyllotoxin, and5-fluorouracil.
 12. The method of claim 11, wherein the taxane ispaclitaxel or docetaxel.
 13. The method of claim 1, wherein treating thepancreatic tumor comprises inducing apoptosis in a pancreatic tumorcell.